Rat Model Of Chronic Constriction Injury Research Articles

Silencing of secreted phosphoprotein 1 attenuates sciatic nerve injury-induced neuropathic pain: Regulating extracellular signal-regulated kinase and neuroinflammatory signaling pathways.

Neuropathic pain (NP) is a chronic pathological pain that affects the quality of life and is a huge medical burden for affected patients. In this study, we aimed to explore the effects of secreted phosphoprotein 1 (SPP1) on NP. We established a chronic constriction injury (CCI) rat model, knocked down SPP1 via an intrathecal injection, and/or activated the extracellular signal-regulated kinase (ERK) pathway with insulin-like growth factor 1 (IGF-1) treatment. Pain behaviors, including paw withdrawal threshold (PWT), paw withdrawal latency (PWL), lifting number, and frequency, were assessed. After sacrificing rats, the L4-L5 dorsal root ganglion was collected. Then, SPP1 levels were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β were determined using qPCR and enzyme-linked immunosorbent assay. The levels of ERK pathway factors were determined via western blot analysis. We found that CCI decreased PWT and PWL, increased the lifting number and frequency, and upregulated SPP1 levels. The loss of SPP1 reversed these CCI-induced effects. Additionally, CCI upregulated IL-1β, TNF-α, IL-6, EGF, and VEGF levels, downregulated TGF-β levels, and activated the ERK pathway, while silencing of SPP1 abrogated these CCI-induced effects. Moreover, IGF-1 treatment reversed the effects of SPP1 loss. The data indicate that silencing SPP1 attenuates NP via inactivation of the ERK pathway, suggesting that SPP1 may be a promising target for NP treatment.

(+)-Catechin Alleviates CCI-Induced Neuropathic Pain in Rats by Modulating the IL34/CSFIR Axis and Attenuating the Schwann Cell-Macrophage Cascade Response in the DRG.

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats.

Potential anxiolytic and antidepressant-like effects of luteolin in a chronic constriction injury rat model of neuropathic pain: Role of oxidative stress, neurotrophins, and inflammatory factors.

This study aimed to examine the effects of luteolin (LUT) on chronic neuropathic pain (NP)-induced mood disorders (i.e., anxiety and depression) by regulating oxidative stress, neurotrophic factors (NFs), and neuroinflammation. Chronic constrictive injury (CCI) was used to induce NP in the animals. Animals in the treatment groups received LUT in three doses of 10, 25, and 50mg/kg for 21days. The severity of pain and mood disorders were examined. Finally, animals were sacrificed, and their brain tissue was used for molecular and histopathological studies. CCI led to cold allodynia and thermal hyperalgesia. Mood alterations were proven in the CCI group, according to the behavioral tests. Levels of glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), B-cell lymphoma-2 (Bcl2), superoxide dismutase (SOD), catalase (CAT), and nuclear factor erythroid-2-related factor 2 (Nrf2) were reduced in the hippocampus (HPC) and prefrontal cortex (PFC). Furthermore, the levels of MDA, Bcl-2-associatedXprotein (Bax), and inflammatory markers, including nuclear factor kappa B (NF-κB), NLR family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), IL-18, IL-6, and tumor necrosis factor-α (TNF-α) significantly increased in the HPC and PFC following CCI induction. LUT treatment reversed the behavioral alterations via regulation of oxidative stress, neurotrophines, and inflammatory mediators in the HPC and PFC. Findings confirmed the potency of LUT in the improvement of chronic pain-induced anxiety- and depressive-like symptoms, probably through antioxidant, anti-inflammatory, and neuroprotective properties in the HPC and PFC.

Duhuo Jisheng decoction alleviates neuroinflammation and neuropathic pain by suppressing microglial M1 polarization: a network pharmacology research

BackgroundNeuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear.MethodsUsing network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia.ResultsOur screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial M1 polarization to improve neuroinflammation, PPARG may have been involved in the process.ConclusionThe multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP.

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model.

Fu's subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal's body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat's discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

DEAD-box helicase 54 regulates microglial inflammatory response in rats with chronic constriction injuries through NF-κB/NLRP3 signaling axis.

Neuropathic pain (NP) is caused by damage to or disease of the somatosensory nervous system, but its mechanism is still not fully understood. In this study, DEAD-box helicase 54 (DDX54) was targeted, and its regulatory role was explored in a chronic constriction injury (CCI) rat model. Microglia and HMC3 cells were stimulated with LPS. The interaction between DDX54 and myeloid differentiation factor-88 adapter protein (MYD88) was verified. A CCI of sciatic nerve model in rats was established. Behavioral testing was performed before and after the CCI. The expressions of IL-1β, TNF-α, and IL-6 were upregulated, and those of DDX54, MYD88, NF-κB, and NOD-like receptor 3 (NLRP3) were upregulated in microglia and HMC3 cells after LPS induction. DDX54 knockdown in microglia and HMC3 cells inhibited IL-1β, TNF-α, and IL-6 expressions and downregulated the protein levels of MYD88, p-NF-κB p65 (p-p65), and NLRP3. DDX54 overexpression promoted the stability of MYD88 mRNA. DDX54 binds to the MYD88-3'-untranslated region (UTR). DDX54 interference in rats could alleviate the decrease of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) induced by CCI, inhibit Iba1 expression, and reduce inflammatory factors as well as MYD88 and NF-κB expressions. DDX54 promotes the activation of NF-κB/NLRP3 signaling by regulating MYD88 mRNA stability, thereby affecting inflammatory response and NP progression in CCI rats.NEW & NOTEWORTHY The role of DDX54 protein in LPS-induced microglia and a chronic constriction injury (CCI) rat model was investigated for the first time. DDX54 interference can inhibit microglial activation and reduce the secretion of inflammatory factors. The interaction between DDX54 protein and MYD88 mRNA was explored for the first time. DDX54 promotes NF-κB/NLRP3 signaling activation by regulating MYD88 transcription in a CCI rat model.

Analgesic effect of safinamide mesylate in a rat model of neuropathic pain

Pain is one of the most frequent non-motor symptoms of Parkinson’s disease (PD). Neuropathic pain is highly prevalent in PD and negatively affects the quality of life of patients with PD. However, there is currently no evidence-based treatment for its control. Safinamide, a monoamine oxidase (MAO)-B inhibitor with a sodium channel inhibitory effect, showed improvement in PD-related pain in several clinical trials. However, it is unclear for which of the various types of pain in PD safinamide is effective. The aim of the present study was to examine the effect of safinamide on neuropathic pain in a rat model of chronic constriction injury (CCI). Pain was evaluated on postoperative days 14 and 21 using von Frey or weight-bearing tests. Male CCI model rats showed a decreased paw withdrawal threshold and a weight-bearing deficit on postoperative days 14 and 21. Single oral administration of safinamide (15, 30, 45 or 70 mg/kg) dose-dependently improved neuropathic pain in both pain assessments on day 14. Subsequently, the 15 and 45 mg/kg dose groups were administered safinamide orally once daily until day 21. With repeated administration, the effect of safinamide on pain was enhanced. The present findings show that safinamide improves neuropathic pain in male CCI model rats. Further animal model research and pathological and molecular pharmacological investigations are warranted.

MG53 ameliorates nerve injury induced neuropathic pain through the regulation of Nrf2/HO-1 signaling in rats

Neuropathic pain is one of the most common types of chronic pain, and it arises as a direct consequence of a lesion or disease that affects the somatosensory system. Mitsugumin53 (MG53), which is a member of the TRIM family of proteins and is known as TRIM72, exerts protective effects on muscle, lung, kidney, brain, and other cells or tissues. Recently, increasing evidence has indicated that MG53 plays a vital role in regulating neuroinflammation and oxidative stress. However, the relationship between MG53 and neuropathic pain is unclear. In this study, we aimed to explore the role of MG3 in neuropathic pain after chronic constriction injury (CCI) to the sciatic nerve in rats. To explore the mechanism of MG53 regulating the development of neuropathic pain, the rats was injected (intrathecal injection) of recombinant human MG53 (rhMG53) protein and/or nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA after CCI. Mechanical allodynia or thermal hyperalgesia was assessed by the 50% paw withdrawal threshold (PWT) or the paw withdrawal latency (PWL). The target molecules was detected using western blotting (WB), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), biochemical evaluations, and Dihydroethidium (DHE) staining. The results indicated that the expression level of MG53 in the spinal cord was increased after CCI in rats. Moreover, intrathecal injection with rhMG53 protein notably alleviated CCI-induced mechanical allodynia, thermal hyperalgesia, neuroinflammation，oxidative stress and the increased level of reactive oxygen species (ROS) via activation of the Nrf2/heme oxygenase-1 (HO-1) signaling pathway. However, administration of Nrf2 siRNA abrogated the analgesic, anti-inflammatory and antioxidant effects of rhMG53 in CCI model rats. Our study demonstrated that MG53 improved neuropathic pain, neuroinflammation, and oxidative stress via activation of the Nrf2/HO-1 signaling pathway in the spinal cord of CCI model rats, which suggested that MG53 may serve as a new target for the treatment of neuropathic pain.

Evaluating the Effects of Probiotic Supplementation on Neuropathic Pain and Oxidative Stress Factors in an Animal Model of Chronic Constriction Injury of the Sciatic Nerve

Introduction: Neuropathic pain is a common and painful somatosensory nervous system disease, and its treatment remains a medical challenge. Evidence demonstrates that gut microbiota alters in neuropathic pain and, therefore, improvement of the gut flora may affect the disease. The present study aimed to evaluate the antinociceptive effect of probiotics in neuropathic pain and oxidative biomarkers’ responsiveness to the probiotic treatment. Methods: Using chronic constriction injury (CCI) of the rats' sciatic nerve, neuropathic pain was induced. Investigating the analgesic effect of the probiotics mixture, 40 male rats were randomly assigned to 4 groups (n=10 for each): Sham-operated (SM), and CCI model rats orally received 1 mL saline (CS), or 100 mg/kg gabapentin (CG) or 1 mL probiotics mixture (CP) Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium bifidum (109 CFU of each) daily. Using behavioral tests, the pain was assessed on days 1, 4, 7, 14, and 21 of the study. Finally, the biochemical evaluation of sciatic nerve tissue was done. Results: Probiotics decreased cold and mechanic allodynia and thermal hyperalgesia. Reducing lipid peroxidation levels and increasing total antioxidant capacity, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were also significant in the probiotics group. Conclusion: These findings suggest that probiotics have analgesic effects on the CCI model of neuropathic pain via increasing the antioxidant capacity of the rats’ sciatic nerve.

Eriocalyxin B inhibits inflammation induced by CCI-induced microglia activation to relieve neuropathic pain through inhibition of JAK2/STAT3 and NF-κB pathways

Neuropathic pain is a very troublesome disease that seriously affects human life. Eriocalyxin B (EriB) has been revealed to attenuate various diseases through its anti-inflammatory effects, but its regulatory effects on neuropathic pain remains unclear. The paw withdrawal threshold and paw withdrawal thermal latency were detected through mechanical allodynia and thermal hyperalgesia tests. The spinal injury was assessed through hematoxylin and eosin staining. The cell apoptosis was measured through terminal deoxynucleotide transferase-mediated dUTP nick end-labeling assay. The protein expressions were examined through Western blot analysis. The mRNA expression was examined through reverse transcription-quantitative polymerase chain reaction. The ionized calcium-binding adaptor molecule 1 level in the spinal cord was evaluated through immunofluorescence assay. The levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were measured through enzyme-linked-immunosorbent serologic assay. The chronic constriction injury (CCI) rat model was constructed for the study. Our results demonstrated that EriB relieved CCI-stimulated neuropathic pain and nerve damage. In addition, the enhanced neural apoptosis mediated by CCI induction was reduced after EriB treatment. In addition, EriB inhibited CCI-induced microglia activity and inflammation. At last, the Janus kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor kappa B (NF-κB) pathways were activated in CCI rat model, which were attenuated following EriB treatment. Importantly, EriB (10 mg/kg) had a strong effect that was similar to the positive control (1-μg/kg dexmedetomidine), suggesting that EriB may be an effective drug for neuropathic pain. This study demonstrated that EriB inhibited inflammation caused by CCI-induced microglia activation to relieve neuropathic pain through inhibition of JAK2/STAT3 and NF-κB pathways. This study may highlight the regulatory functions of EriB in the treatment of neuropathic pain.

Wet cupping therapy increases the time withdrawal latency (TWL) and decreases GABA-A receptor expression in the spinal cord in a rat model of neuropathic pain

Background & Objective: Neuropathic pain (NP) is a pain due to a somatosensory lesion. NP leads to disruption of health, work, social relationships, hobbies, sleep, mood, and cognitive function. Up till now, the treatment of NP remains unsatisfactory. It makes many patients seek alternative therapies, including wet cupping therapy (WCT). We aimed to analyze the effects of WCT against NP in chronic constriction injury (CCI) in a rat model by assessing the increase in time withdrawal latency (TWL) and GABA-A receptors expression in the spinal cord.
 Methodology: We used CCI as one of the NP models in Rattus norvegicus species of rats and treated with WCT. We used 21 four months old male rats, divided into 3 groups (n = 7); P1 (sham CCI group), P2 (CCI group), and P3 (CCI plus WCT group). WCT was given two times every week for three weeks with 5 min of first cupping using −200 mmHg, and followed by ten punctures and then 5 min of second cupping using −200 mmHg. TWL assesment was conducted by using hotplate. Expresion of GABA-A receptor was evaluated with immunohistochemistry.
 Results: WCT significantly increased TWL with P = 0.0001 and decreased expresion of GABA-A receptors in the spinal cord of CCI rat model.
 Conclusion: This research concluded that wet cupping therapy could increase time withdrawal latency and decrease the expression of GABA-A receptors in the spinal cord in chronic constriction injury rat model. This result suggests a promising method in controlling neuropathic pain. However, further investigations are required to understand the mechanism.
 Abbreviations: CCI: Chronic Constriction Injury; CT: Cupping Therapy; DCT: Dry Cupping Therapy; NP: Neuropathic Pain; TWL: Time Withdrawal Latency; WCT: Wet Cupping Therapy;
 Key words: Chronic constriction injury; CCI rat model; GABA-A receptors; Neuropathic pain; Pain; Time withdrawal latency; TWL; Wet cupping therapy.
 Citation: Hidayati HB, Qurnianingsih E, Widjiati, Khaerunnisa S, Puspamaniar VA, Susetyo RD. Wet cupping therapy increases the time withdrawal latency (TWL) and decreases GABA-A receptor expression in the spinal cord in a rat model of neuropathic pain. Anaesth. pain intensive care 2022;27(1):97−103; DOI: 10.35975/apic.v27i1.2124
 Received: July 05, 2022; Reviewed: July 08,2022; Accepted: December 12, 2022

Effects of intrathecally administered interferon α on chronic constriction injury model rats' mechanical pain threshold and G protein expression in the spinal cord.

The aim of the study was to explore the analgesic mechanism of effects of intrathecally administered interferon a (IFN-a) on chronic constriction injury (CCI) model rats. 24 rats were divided into 6 groups, with 4 rats in each group, including the negative control group (Group N, no operation or treatment), the sham operation group (Group S, only the left sciatic nerve of the rats was exposed without ligation, 0.9% NaCl was intrathecally administered), and four experimental groups (CCI model was established first and then different drugs were intrathecally administered respectively), including 0.9% NaCl (Group C), IFN-a (Group CI), morphine (Group CM), and IFN-a combined with morphine (Group CIM). The mRNA levels of G proteins in both the spinal cord and dorsal root ganglia (DRG), as well as the content of amino acid and chemokine (C-X-C motif) ligand 6 (CXCL-6) in the cerebrospinal fluid were measured and analysed in each group. Intrathecal administration of IFN-a increased the mechanical pain threshold in CCI rats (33.32 ±1.36 vs. 21.08 ±1.59, p < 0.001), achieving the effect comparable to that of morphine (33.32 ±1.36 vs. 32.44 ±3.18, p > 0.05), increased the mRNA expression level of Gi protein (0.62 ±0.04 vs. 0.49 ±0.05, p = 0.006), and decreased the mRNA expression level of Gs protein in the spinal cord (1.80 ±0.16 vs. 2.06 ±0.15, p = 0.035) and DRG (2.11 ±0.10 vs. 2.79 ±0.13, p < 0.001). The intrathecal administration of both IFN-a and morphine can reduce the glutamate content in the cerebrospinal fluid (261.55 ±38.12 vs. 347.70 ±40.69, p = 0.012), but without any statistically significant difference in the content of CXCL-6 across all groups ( p > 0.05). Intrathecal injection of IFN-a improved the mechanical pain threshold in CCI rats, so we inferred that intrathecal administration of IFN-a had analgesic effects on neuropathic pain, possibly related to the activation of G-proteincoupled µ receptors in the spinal cord and the inhibition of glutamate release.

LncRNA FTX ameliorates neuropathic pain by targeting miR-320a in a rat model of chronic constriction injury.

Long non-coding RNAs (lncRNAs) participate in the process of neuropathic pain (NP). Herein, the goal of this research was to examine the roles of lncRNA five prime to XIST (FTX) in influencing chronic constriction injury (CCI)-induced NP. We have established a rat CCI model to simulate NP in vivo. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect mRNA levels of FTX, microRNA (miR)-320a, and runt-related transcription factor 2 (RUNX2) in the spinal cord. This was followed by subsequent regulation of FTX or miR-320a levels in vivo by intrathecal injection of overexpression FTX or miR-320a mimic lentivirus. The behaviour of rat NP the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of pro-inflammatory and anti-inflammatory factors in the spinal cord tissue. A correlation between FTX and miR-320a, and RUNX2 was validated by luciferase reporter. FTX levels were reduced in CCI rats ( p < 0.05), and miR-320a was a direct target of FTX. Overexpression of FTX typically reduced PWL and PWT as well as neuroinflammation thus alleviating NP ( p < 0.05). However, increasing miR-320a reversed the alleviation of FTX on NP, increased PWL and PWT, and promoted neuroinflammation ( p < 0.05). Additionally, RUNX2, which is a miR-320a target gene, was significantly repressed in CCI rats and its expression was increased by FTX, however, this increase was attenuated by elevated miR-320a ( p < 0.05). In the CCI-induced NP rat model, FTX attenuates NP and neuroinflammation by regulating the miR-320a/RUNX2 axis. This provides a new vision for NP treatment.

The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain.

Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic-lysosomal pathway in the rat CCI model. Sprague-Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.

Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

BackgroundGrowing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear.MethodsA chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPβ-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro.ResultsSignificant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPβ, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPβ knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector.ConclusionsThis pre-clinical investigation reveals that C/EBPβ-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.

Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor.

Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1β. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.

Electroacupuncture Suppresses CCI-Induced Neuropathic Pain through GABAA Receptors.

Neuropathic pain remains a chronic and intractable pain. Recent studies have shown a close relationship between gamma-aminobutyric acid A (GABAA) receptor and neuropathic pain. Spinal cord GABAA receptors are key modulators of pain processing. Electroacupuncture (EA) is currently used worldwide to relieve pain. The immunomodulatory effect of EA in animals has been proposed in previous studies. However, it remains unclear how EA contributes to alleviating neuropathic pain. In this study, the chronic constriction injury (CCI) rat model was used to explore the relationship between GABAA receptor and neuropathic pain. We also investigated whether EA treatment could ameliorate pain hypersensitivity by modulating the GABAA receptor. To determine the function of EA in neurological diseases, in this study, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed to determine the threshold of pain. In addition, we used Western blot, immunofluorescence, and real-time quantitative PCR to confirm whether EA treatment relieves pain hypersensitivity by regulating GABAA receptors. The morphology of synapse was examined using an electron microscope. In the present study, EA relieved mechanical allodynia and thermal hyperalgesia. EA also inhibited microglial activation in the spinal cord, accompanied by increased levels of GABAARα2, GABAARα3, and GABAARγ2 subunits. However, the analgesic effect of EA was attenuated by treatment with the GABAA receptor antagonist bicuculine. Overall, the present results indicate that microglia and GABAA receptor might participate in EA analgesia. These results contribute to our understanding of the impact of EA on rats after sciatic nerve compression, providing a theoretical basis for the clinical application of EA analgesia.

MiR-204-5p Alleviates Neuropathic Pain by Targeting BRD4 in a Rat Chronic Constrictive Injury Model.

PurposeThe pathogenesis of neuropathic pain is complex, and previous studies have found that microRNAs are important regulators of neuropathic pain and are associated with the progression of neuropathic pain. This study aims to explore the level and role of miR-204-5p in the chronic constrictive injury (CCI) model of rats.Patients and MethodsThe CCI rat model was constructed to evaluate paw withdrawal threshold (PWT), paw withdrawal latency (PWL), the expressions of miR-204-5p, and the contents of inflammatory factors in the model. Overexpression of miR-204-5p in rat spinal cord was induced by intrathecal injection of miR-204-5p mimics. PWT and PWL were used to estimate mechanical and thermal pain thresholds. IL-6 and TNF-α were determined by ELISA. Luciferase reporter gene was conducted to verify the targeting relationship between miR-204-5p and BRD4.ResultsmiR-204-5p was abnormally down-regulated in the CCI group. The thresholds of mechanical and thermal pain stimulation in the CCI group were lower, and the levels of inflammatory factors were higher than those in the sham group. Overexpression of miR-204-5p alleviated PWT, PWL and inflammatory factors. Besides, the luciferase reporter gene showed that BRD4 was a target gene of miR-204-5p.ConclusionThese results suggested that miR-204-5p may alleviate neuropathic pain and inflammation through targeted regulation of BRD4 expression.

Co-Combination of Pregabalin and Withaniacoagulans-Extract-Loaded Topical Gel Alleviates Allodynia and Hyperalgesia in the Chronic Sciatic Nerve Constriction Injury for Neuropathic Pain in Animal Model.

The current study reports the fabrication of co-combination gel using Pregabalin and Withania coagulans fruit extract to validate its effectiveness for neuropathic pain in chronic constriction injury (CCI) rat models. Three topical gels were prepared using Carbopol 934 through a pseudo-ternary phase diagram incorporating the Pregabalin (2.5%), Withania coagulans extract (2%), and co-combination of both Pregabalin (2.5%) and Withania coagulans extract (2%). Gels were characterized. FTIR showed a successful polymeric network of the gel without any interaction. The drug distribution at the molecular level was confirmed by XRD. The AFM images topographically indicated the rough surface of gels with a size range from 0.25 to 330 nm. DSC showed the disappearance of sharp peaks of the drug and extract, showing successful incorporation into the polymeric network of gels. The in vitro drug release of co-combination gel was 73% over 48 h. The mechanism of drug release by combination gel was Higuchi+ fickian with values of n (0.282) and R2 (0.947). An in vivo study for pain assessment via four methods: (i) heat hyperalgesia, (ii) cold allodynia, (iii) mechano-hyperalgesia, and (iv) dynamic mechano-allodynia, confirmed that topical treatment with co-combination gel reduced the pain significantly as indicated by the p value: R1 (p < 0.001), R2 (p < 0.001), R3 (p < 0.015), and R4 (p < 0.0344). The significance order was R2 (****) > R1 (***) > R3 (**) > R4 (*) > R5 (ns).

Preventive Intrathecal Injection of Bupivacaine Alleviated Microglia Activation and Neuropathic Pain in a Rat Model of Chronic Constriction Injury

Spinal microglia are crucial to neuronal hyper-excitability and pain hypersensitivity. The local anesthetic bupivacaine is commonly used for both peripheral and spinal anesthesia. The pain-relief effects resulting from the peripheral and systemic administration of bupivacaine have been previously reported. In this study, the preventive effects of intrathecal bupivacaine administration against neuropathic pain were revealed in a rat model of sciatic nerve chronic constriction injury (CCI). Using a CCI rat model, pain hypersensitivity, characterized by mechanical allodynia and thermal hyperalgesia, correlated well with microglia M1 polarization, activation and pro-inflammatory cytokine expression in both spinal cord dorsal horns and sciatic nerves. Bupivacaine attenuated pain behaviors and inflammatory alternations. We further identified that the Interferon Regulatory Factor 5 (IRF5)/P2X Purinoceptor 4 (P2X4R) and High Mobility Group Box 1 (HMGB1)/Toll-Like Receptor 4 (TLR4)/NF-κB inflammatory axes may each play pivotal roles in the acquisition of microglia M1 polarization and pro-inflammatory cytokine expression under CCI insult. The relief of pain paralleled with the suppression of microglia M1 polarization, elevation of microglia M2 polarization, and inhibition of IRF5/P2X4R and HMGB1/TLR4/NF-κB in both the spinal cord dorsal horns and sciatic nerve. Our findings provide molecular and biochemical evidence for the anti-neuropathic effect of preventive bupivacaine.